Abstract
Temporal separation of red (R) and blue (B) (alternating R/B) photons has been reported to increase leaf area, photon capture, and yield of lettuce compared with delivering both colors together (concurrent R+B). We grew three diverse lettuce cultivars (Grand Rapids, Rex, and Red Sails) under concurrent R+B photons (9/1 ratio) and alternating R/B photons (9/1 ratio) under an equal daily light integral (DLI) of either 8.6 or 23 mol⋅m−2⋅d−1. Contrary to five previous studies, we found no increase in either leaf area or fresh mass and dry mass in any of the alternating R/B photon treatments compared with concurrent R+B photons. In fact, at a DLI at 8.6 mol⋅m−2⋅d−1, alternating R/B photons decreased the dry mass of ‘Grand Rapids’ and ‘Rex’ lettuce by 38% and 17%, respectively. Two previous studies reported that photosynthetic rates increased with alternating R/B photons; however, we found that the net assimilation rate was generally decreased by alternating R/B photons. An analysis of images obtained from automated digital photography revealed that the relative expansion rate of leaves was 61% higher during intervals of pure B rather than intervals of pure R photons at the same photosynthetic photon flux density; however, this did not result in a higher leaf area compared with concurrent R+B photons. Overall, our studies do not indicate that alternating R/B photons increase lettuce leaf area or yield compared with concurrent R+B photons.
Light is both an energy source for photosynthesis and an information source regarding the surrounding environment. In controlled environment agriculture, light-emitting diodes (LEDs) are often used in crop production under sole-source lighting because of their low heat production, great controllability, and high efficacy that converts energy to light output (Cammarisano et al. 2021; Kusuma et al. 2020; Nelson and Bugbee 2014). Crops grown under LED are often exposed to light regimes that are very different from sunlight because of the narrow spectral distribution and controllability of LEDs. Therefore, it is difficult to predict the morphological and physiological responses of crops to LED (Graham et al. 2019). The use of LEDs offers opportunities to study the signaling mechanism of plants and fine-tune the physiological and morphological responses of the crop to increase profitability for growers.
Multiple studies have reported that alternating red (R) photons and blue (B) photons (alternating R/B photons) could increase lettuce yield compared with concurrent R and B photons (concurrent R+B photons) under sole-source lighting. Ohtake et al. (2018) found that ‘Summer Surge’ lettuce, when grown under alternating 12-h R photons at 120 μmol⋅m−2⋅s−1 and 12-h B photons at 40 μmol⋅m−2⋅s−1, accumulated 20.2% higher fresh weight and 18.9% higher dry mass than plants grown under concurrent R and B photons despite the same daily light integral (DLI) of 6.9 mol⋅m−2⋅d−1 (Fig. 1). A follow-up study performed by the same group reported the following similar results: plants grown under alternating monochromatic photons had 22.2% higher fresh weight and 13.3% higher dry mass than plants grown under concurrent R+B photons (Ohtake et al. 2021) (Fig. 1). A larger projected canopy size was reported for lettuce plants grown under alternating R/B photons (Ohtake et al. 2021). These results indicated that a larger canopy size and, thus, higher photon capture of lettuce grown under alternating photon flux were the underlying reasons for enhanced growth.
Summary of previous studies of the effect of alternating red (R)/blue (B) and concurrent R+B photons on fresh mass and dry mass of lettuce. The third column presents the light treatment in the previous literature. The fifth and sixth columns show the change in the fresh weight and dry mass relative to the control treatment in each study. ns = not statistically different from the control.
Citation: HortScience 59, 7; 10.21273/HORTSCI17720-24
The highest increase in yield from alternating photon color was reported for ‘Greenwave’ lettuce (Kuno et al. 2017) (Fig. 1). Plants grown under alternating 12-h B photons and 12-h R photons, both at 120 μmol⋅m−2⋅s−1, accumulated 62.0% and 44.4% more shoot fresh mass and dry mass, respectively, than plants grown under concurrent 60 μmol⋅m−2⋅s−1 B and 60 μmol⋅m−2⋅s−1 R photons with a 24-h photoperiod (Kuno et al. 2017). Interestingly, when grown under alternating R and B photons at 100 μmol⋅m−2⋅s−1 in 12-h intervals, ‘Greenwave’ lettuce plants tended to have a greater increase in shoot fresh weight than shoot dry mass compared with lettuce plants grown under concurrent 50 μmol⋅m−2⋅s−1 B and 50 μmol⋅m−2⋅s−1 R photons (Masuda et al. 2021; Takasu et al. 2019) (Fig. 1). Plants grown under alternating R/B photons resulted in 47.4% and 23.4% higher shoot fresh mass than plants grown under concurrent R + B photons, without a statistically significant increase in dry mass, in two studies of ‘Greenwave’ lettuce for better readability (Masuda et al. 2021; Takasu et al. 2019) (Fig. 1). A multiple regression analysis suggested that the increase in total leaf area under alternating R/B photons was related to the increase in shoot fresh weight of ‘Greenwave’ lettuce (Masuda et al. 2021), similar to the results observed for ‘Summer surge’ lettuce.
The mechanism by which alternating R/B photons increased leaf and canopy expansion is not clear. The previous studies hypothesized that R and B photons induced antagonistic signals on crop growth, and that temporally separating R and B radiation could “resolve the conflict” and result in more efficient growth (Chen et al. 2017; Masuda et al. 2021; Ohtake et al. 2021). However, after millions of years of evolution, one would expect that plants would adapt to the sunlight spectrum and achieve the highest growth under such a spectrum. Recent research reported that pure B photons promote stem and leaf elongation compared with pure R photons and concurrent R and B photons in several species (Hata et al. 2013; Hernández and Kubota 2016; Hirai et al. 2006; Johnson et al. 2020; Kim et al. 2014; Kong et al. 2018, 2019; Spalholz et al. 2020). In cucumber and microgreens, the stem and leaf elongation translated into higher shoot dry mass under pure B light compared with R or concurrent R and B light treatments (Hernández and Kubota 2016; Johnson et al. 2020; Kong et al. 2020) It is possible that under alternating R/B photons, leaf expansion was greater during the intervals of pure B photons than under concurrent R+B photons.
A difference in photosynthesis was also noted for lettuce grown under alternating R/B photons and concurrent R+B photons. The higher net assimilation rate (NAR) and leaf photosynthetic rates (Pn) of lettuce grown under alternating R/B photons than those under concurrent R+B photons were also reported (Ohtake et al. 2018; Takasu et al. 2019). At the leaf level, Pn directly quantifies the short-term photosynthetic rate of single leaves. When measured under 50 μmol⋅m−2⋅s−1 R and 50 μmol⋅m−2⋅s−1 B light, plants grown under alternating R/B photons had higher leaf Pn than plants grown under concurrent R+B photons (Takasu et al. 2019). The daily carbon gain, which quantifies the accumulation of net carbon assimilation over 1 d, was calculated based on the leaf Pn measured in situ. In contrast to the higher leaf Pn reported previously, the daily carbon gain was similar between plants grown under alternating R/B and concurrent R+B radiation (Ohtake et al. 2018, 2021), which implied that photosynthetic rates were not different and did not contribute to the difference in yield. At the whole plant level, NAR is used to quantify net photosynthetic efficiency (photosynthesis minus respiration) per leaf area. The NAR is calculated as the rate of leaf dry mass production per unit of leaf area. When integrated over the whole growing cycle, NAR can be calculated as the shoot dry mass divided by the total leaf area (g⋅m−2 of leaf area) (Snowden et al. 2016). Ohtake et al. (2018) found that the NAR of lettuce plants grown under alternating R/B radiation was higher than that of plants grown under concurrent R+B radiation at 6 to 12 d after light treatment started; however, the difference disappeared at a later growth stage. Collectively, it is unclear whether alternating R/B might increase the photosynthetic rate.
Our objective was to more rigorously quantify the leaf expansion, photosynthesis, and yield of lettuce plants grown under concurrent R+B and alternating R/B photons. We hypothesized that alternating R/B photons would increase leaf expansion compared with concurrent R+B photons, which would lead to higher yield. We also hypothesized that alternating R/B photons would increase the photosynthetic rate and further contribute to higher yield than concurrent R+B photons.
Materials and Methods
Plant material and growth condition.
Three diverse lettuce cultivars were selected for this study: Grand Rapids (Mountain Valley Seed Co., Salt Lake City, UT, USA), Rex, and Red Sails (Johnny’s Selected Seeds, Winslow, ME, USA). Grand Rapid is a green leaf lettuce similar to the cultivars used in previous studies. ‘Rex’ is a butterhead (Boston) type of lettuce with a tight canopy architecture. ‘Red Sails’ is a red lettuce with high levels of anthocyanins.
Lettuce seeds were sowed in 10-cm square pots filled with peat-based substrate and germinated under their respective light treatments. Six days after sowing, seedlings were thinned to one plant per pot. Four plants of each cultivar were used per treatment. Fertigation was supplied with drip emitters attached to each pot, similar to those described by Westmoreland et al. (2021). The fertilizer solution was made with a complete liquid fertilizer [Peter’s Peat-lite professional 20–10–20 (20N–4.4P–16.6K); Everris NA, Dublin, OH, USA] at a rate of 120 mg⋅L−1 nitrogen in solution. AgSil 16H (PQ Corporation, Malvern, PA, USA) was added using a second proportioner for the liquid fertilizer at a rate of 8.4 mg silicon (0.3 mmol silicon) per liter. Fertigation was provided every other day at the seedling stage, and then daily after seedling stage. The electrical conductivity (EC) and pH of fertilizer solution were measured weekly and were 0.13 ± 0.03 S⋅m−1 and 7.1 ± 0.4 (average ± SD), respectively. The air temperature and vapor pressure deficit were recorded by a temperature and humidity sensor (HMP45A; Campbell Scientific, Logan, UT, USA) every 5 min and were 22.1 ± 0.9 °C and 1.25 ± 0.24 kPa (average ± SD) throughout this study. The CO2 concentration in the grow room was 1186 ± 117 μmol⋅mol−1 (average ± SD), which was measured by an infrared gas analyzer (Li850; LI-COR Biosciences, Lincoln, NE, USA) every 10 s.
Light treatments.
The grow room was divided into five sections that can be independently treated with lights of different spectrums, intensity, and photoperiods, similar to what was described by Westmoreland et al. (2021). The R and B LED fixtures (Fluence Bioengineering, Austin, TX, USA) were used. The B LEDs peaked at 442 nm with full width at half maximum of 19 nm. Red LEDs peak at 663 nm with full width at half maximum of 21 nm. The photosynthetic photon flux density (PPFD) and spectral distribution of light treatments were measured with a spectroradiometer (PS-300; Apogee, Logan, UT, USA) at approximately 90 mm above the substrate level. For the alternating R/B radiation treatments, R and B photons were applied in 12-h intervals, from 00:00 to 12:00 and from 12:00 to 00:00, and the order was randomly assigned for each treatment.
At the DLI of 23.04 mol⋅m−2⋅d−1, one alternating R/B photon treatment was applied, with alternating between R photons at 480 μmol⋅m−2⋅s−1 and B photons at 53.3 μmol⋅m−2⋅s−1. Two concurrent R+B photon treatments were applied. The first concurrent R+B light had a photoperiod of 24 h, consisting of 240 μmol⋅m−2⋅s−1 R photons and 26.7 μmol⋅m−2⋅s−1 B photons. The second concurrent R+B light had a photoperiod of 16 h, consisting of 360 μmol⋅m−2⋅s−1 R photons and 40 μmol⋅m−2⋅s−1 B photons (Fig. 2). All treatments had the same DLI of R and B photons and a ratio of R photons to B photons (R:B ratio) of 9:1. This study was performed three times, except for the concurrent R+B photon treatment with a photoperiod of 16-h, which missed one replicate.
Summary of treatments in the current study of the effects of alternating red (R)/blue (B) and concurrent R+B photons on fresh mass and dry mass of lettuce.
Citation: HortScience 59, 7; 10.21273/HORTSCI17720-24
We followed these studies with a study using a lower DLI of 8.6 mol⋅m−2⋅d−1 (Fig. 2). We tested two alternating R/B photon treatments under this DLI. The first alternating R/B photon treatment alternated between 180 μmol⋅m−2⋅s−1 R photons and 20 μmol⋅m−2⋅s−1 B photons. The second alternating R/B photon treatment alternated between 100 μmol⋅m−2⋅s−1 R photons and 100 μmol⋅m−2⋅s−1 B photons, which had a different R:B ratio than all other treatments. One concurrent R+B photon treatment was used as the control, with 24 h of 90 μmol⋅m−2⋅s−1 R and 10 μmol⋅m−2⋅s−1 B photons (Fig. 2). Four plants of each of the three cultivars were grown in separate containers, which were arranged in a completely random design. The PPFD of R and B photons for each plant were individually controlled by adjusting the distance between plant and lights. The light conditions were measured individually for each plant (Supplementary Table 1).
All the light treatments are summarized in Fig. 2. Low DLI treatments had a DLI similar to that reported in the previous literature. The light treatment started at sowing and continued until harvest at 28 d.
Data Collection
Leaf gas exchange.
Gas exchange measurements were performed 20 d after sowing using a portable gas exchange system (Li-6800; LI-COR). The Pn of a recently fully expanded leaf that was exposed to light was measured for each plant. Light for measuring Pn was provided by a fluorometer attachment of the Li-6800 (Li-6800-01A; LI-COR), mimicking the light conditions in situ. For example, in the treatment that alternated between 12-h 180 μmol⋅m−2⋅s−1 R photons and 12-h 20 μmol⋅m−2⋅s−1 B photons, leaf Pn was measured under 180 μmol⋅m−2⋅s−1 R photons during the interval of pure R photons in the growth room, and it was measured again under 20 μmol⋅m−2⋅s−1 B photons during the interval of pure B photons in the growth room. The leaf Pn of plants that were grown under concurrent R+B radiation was measured once because we assumed that leaf Pn did not change under constant PPFD and light color. In the measuring chamber, the CO2 concentration, temperature, and vapor pressure deficit were 1200 ± 0 μmol⋅m−2⋅s−1, 23.7 ± 1.2 °C, and 1.2 ± 0.1 kPa, respectively, which were similar to those of the grow room.
Image analysis.
Top-view photos of lettuce plants grown under a DLI of 8.6 mol⋅m−2⋅d−1 were obtained using a Raspberry Pi 4 Model B equipped with a NoIR Camera Module V2 (Raspberry Pi Ltd., Cambridge, UK). The cameras were equipped with long pass filters (SCHOTT RG9; Edmund Optics, Barrington, NJ, USA) that blocked most photons <700 nm from reaching the cameras. This allowed for sharper contrast between plants and the background. In this study, photos were taken hourly, but we only used photos from five time points at 12-h intervals to calculate the relative expansion rates (RER). The time points were mid-day at 18 d after sowing and midnight and mid-day at 19 d and 20 d after sowing. The RER was calculated as the difference between the canopy area in 12-h intervals divided by the canopy area at the beginning of the 12-h intervals for each plant. Therefore, we calculated the RER of four 12-h intervals for the alternating R/B treatment (two were under pure R photons and two were under pure B photons). We present the RER under R photons and under B photons for plants in the alternating R/B photon treatment and RER under the red and blue photon mixture for plants in concurrent R+B photon treatment.
Leaf area, fresh mass, and dry mass at harvest.
At harvest (28 d after sowing), top-view photos were obtained by a cellphone (Pixel 4; Google LLC, Mountain View, CA, USA). The fresh weight of shoots was measured. The leaf area of each plant was measured by a leaf area meter (Li-3000; LI-COR). Then, shoots were dried at 80 °C for at least 72 h, and the dry mass of shoots was recorded. The NAR was calculated as the shoot dry mass divided by the leaf area for each plant.
Statistical analysis.
This study used a completely randomized design, with light treatments randomly assigned to the five sections of the grow room. Dry mass, total leaf area, and NAR were analyzed with an analysis of variance (ANOVA) to determine the effects of light treatments using SAS (SAS University Edition; SAS Institute, Cary, NC, USA). For the DLI of 23 mol⋅m−2⋅d−1, the three repeats over time were used as replicates (n = 3). For the DLI of 8.6 mol⋅m−2⋅d−1, four plants of each cultivar were arranged in a completely random design (n = 4).
The RER and leaf Pn of plants grown under the DLI of 8.6 mol⋅m−2⋅d−1 were analyzed with an ANOVA. Data from three cultivars with four replicates each were pooled (n = 12) because there were no significant differences among cultivars.
A regression analysis was performed to quantify the relationship between the leaf Pn and NAR of lettuce plants grown under a DLI of 8.6 mol⋅m−2⋅d−1 (Microsoft Excel; Microsoft, Seattle, WA, USA).
Results
There was no increase in the fresh mass or dry mass in any of the alternating R/B photon treatments under either DLI compared with the concurrent R+B treatments. The top-view photos of lettuce plants grown under a high DLI of 23 mol⋅m−2⋅d−1 (28 d after sowing) are shown in Fig. 3. Under a high DLI, there was a larger leaf area under alternating R/B light than under 16-h concurrent R+B photons (Fig. 4B); however, this increase in leaf area did not translate into higher shoot dry mass (Fig. 4A). Between lettuce grown under alternating R/B photons and 24-h concurrent R+B photons, which had the same photoperiod and DLI, no change in the shoot dry mass or leaf area was found (Fig. 4A and 4B). The NAR, which represents the photosynthetic rate of lettuce plants, was highest under 24-h concurrent R+B photons and 15.9% higher than the NAR of lettuce under alternating R/B photons (Fig. 4C).
Top-view photos of ‘Grand Rapids’, ‘Rex’, and ‘Red Sails’ lettuce plants grown under the three spectral treatments in the second replicate. 16h RB refers to a 16-h photoperiod with concurrent 360 μmol⋅m−2⋅s−1 red (R) photons and 40 μmol⋅m−2⋅s−1 blue (B) photons. 24h RB refers to a 24-h photoperiod with concurrent 240 μmol⋅m−2⋅s−1 R photons and 26.7 μmol⋅m−2⋅s−1 B photons. 12hR/12hB refers to alternating between 12-h R photons at 480 μmol⋅m−2⋅s−1 and 12-h B photons at 53.3 μmol⋅m−2⋅s−1. The daily light integral (DLI) was 23 mol⋅m−2⋅d−1 for all treatments. The age of the plants was 26 d.
Citation: HortScience 59, 7; 10.21273/HORTSCI17720-24
The effects of spectral treatments on shoot fresh weight (A), total leaf area (B), and net assimilation rate (C) of lettuce plants. RB 16h refers to a 16-h photoperiod with concurrent 360 μmol⋅m−2⋅s−1 red (R) photons and 40 μmol⋅m−2⋅s−1 blue (B) photons. RB 24h refers to a 24-h photoperiod with concurrent 240 μmol⋅m−2⋅s−1 R photons and 26.7 μmol⋅m−2⋅s−1 B photons. R 12h/B 12h refers to alternating between 12-h R photons at 480 μmol⋅m−2⋅s−1 and 12-h B photons at 53.3 μmol⋅m−2⋅s−1. The daily light integral (DLI) was 23 mol⋅m−2⋅d−1 for all treatments. Error bars represent the SD (n = 3).
Citation: HortScience 59, 7; 10.21273/HORTSCI17720-24
Under the low DLI at 8.6 mol⋅m−2⋅d−1 (photos shown in Fig. 5), there was an interaction between light treatments and cultivar (Fig. 6). For both ‘Grand Rapids’ and ‘Rex’ lettuce, alternating R/B photons decreased the shoot dry mass compared with concurrent R+B light, with ‘Grand Rapids’ exhibiting a stronger reduction in shoot dry mass than ‘Rex’ (Fig. 6A). The total leaf areas of ‘Grand Rapids’ and ‘Rex’ (Fig. 6B) showed trends similar to that of the shoot dry mass (Fig. 6A), implying that changes in the leaf area and, thus, light interception were likely responsible for the changes in dry mass. ‘Red Sails’ is a red leaf lettuce and did not show any difference in the shoot dry mass and total leaf area with alternating R/B light and concurrent R+B light (Fig. 6A and 6B). Except for that of ‘Rex’, the NAR did not differ among different light treatments (Fig. 6C). Alternating R/B photons decreased NAR by 11.4% in ‘Rex’ lettuce compared with concurrent R+B photons at the same R:B ratio of 9:1 (Fig. 6C). Alternating R/B photons with an R:B ratio of 1:1 resulted in a similar NAR as concurrent R+B photons (Fig. 6C).
Top-view photos of ‘Grand Rapids’, ‘Rex’, and ‘Red Sails’ lettuce plants grown under the three spectral treatments. 24h R90 + B10 refers to a 24-h photoperiod with concurrent 90 μmol⋅m−2⋅s−1 red (R) photons and 10 μmol⋅m−2⋅s−1 blue (B) photons. 12h R180/12h B20 refers to alternating between 12-h R photons at 180 μmol⋅m−2⋅s−1 and 12-h B photons at 20 μmol⋅m−2⋅s−1. 12h R100/12h B100 refers to alternating between 12-h R photons at 100 μmol⋅m−2⋅s−1 and 12-h B photons at 100 μmol⋅m−2⋅s−1. The daily light integral (DLI) was 8.6 mol⋅m−2⋅d−1 for all treatments. The age of the plants was 27 d.
Citation: HortScience 59, 7; 10.21273/HORTSCI17720-24
The effects of spectral treatments on shoot fresh weight (A), total leaf area (B), and net assimilation rate (C) of ‘Grand Rapids’, ‘Rex’, and ‘Red Sails’ lettuce plants. 24h R90 + B10 refers to a 24-h photoperiod with concurrent 90 μmol⋅m−2⋅s−1 red (R) photons and 10 μmol⋅m−2⋅s−1 blue (B) photons. 12h R180/12h B20 refers to alternating between 12-h R photons at 180 μmol⋅m−2⋅s−1 and 12 h B photons at 20 μmol⋅m−2⋅s−1. 12h R100/12h B100 refers to alternating between 12-h R photons at 100 μmol⋅m−2⋅s−1 and 12-h B photons at 100 μmol⋅m−2⋅s−1. The daily light integral (DLI) was 8.6 mol⋅m−2⋅d−1 for all treatments. Error bars represent the SD (n = 4). Plants were harvested 28 d after sowing.
Citation: HortScience 59, 7; 10.21273/HORTSCI17720-24
Effect on the RER.
To further investigate the effect of alternating R/B photons on canopy expansion and light interception, the RER and measured leaf Pn were measured in situ. The RER represents photon capture, which is the first component of overall photon conversion efficiency (Fig. 7). Lettuce plants grown under alternating R/B photons expanded at different rates between the 12-h intervals of different light colors (Fig. 7A). Here, we only present the data of lettuce grown under alternating 100 μmol⋅m−2⋅s−1 R photons/100 μmol⋅m−2⋅s−1 B photons and data from lettuce grown under concurrent 90 μmol⋅m−2⋅s−1 R photons and 10 μmol⋅m−2⋅s−1 B photons because the PPFDs in these treatments were identical. Under 100 μmol⋅m−2⋅s−1 B photons, lettuce expanded 61.2% faster than it did under 100 μmol⋅m−2⋅s−1 R photons (Fig. 7A). Lettuce plants that were grown under concurrent R+B photons did not show any diurnal pattern and had intermediate RER (Fig. 7A). The leaf Pn was significantly different under pure B photons and under R photons, despite the same PPFD (Fig. 7B). However, Pn under concurrent R+B photons was intermediate (Fig. 7B).
The relative expansion rate (RER) of canopies at 19 d after sowing (A) and the leaf level net photosynthetic rates (leaf Pn) (B) of lettuce. Lettuce plants grew under alternating 12-h red (R) photons at 100 μmol⋅m−2⋅s−1 and 12-h blue (B) photons at 100 μmol⋅m−2⋅s−1 (alternating R/B photons) and concurrent 90 μmol⋅m−2⋅s−1 R photons and 10 μmol⋅m−2⋅s−1 B photons (concurrent R+B photons). The RER and leaf Pn of plants in the alternating R/B photon treatment were measured in situ under B photons (B100; blue bars) and under pure R photons (R100; red bars) in alternating R/B photons. Plants in the concurrent R+B photon treatment were measured only once (R90B10; purple bars). The daily light integral (DLI) was 8.6 mol⋅m−2⋅d−1 for all treatments. Error bars represent the SD (n = 12).
Citation: HortScience 59, 7; 10.21273/HORTSCI17720-24
The leaf Pn quantified the instantaneous photosynthetic rate of a 6-cm2 leaf section. The NAR is the efficiency of dry mass production per leaf area of the whole plant and, in our case, incorporates the whole growing cycle. There was a strong correlation between the leaf Pn and NAR of lettuce plants grown under the DLI of 8.6 mol⋅m−2⋅d−1 (r2 = 0.83) (Fig. 8).
The leaf net photosynthetic rates (leaf Pn) measured by a portable gas exchange system (Li-6800) strongly correlated with the net assimilation rate calculated based on the shoot dry mass and leaf area of ‘Grand Rapids’, ‘Rex’, and ‘Red Sails’ lettuce grown under the daily light integral (DLI) of 8.6 mol⋅m−2⋅d−1.
Citation: HortScience 59, 7; 10.21273/HORTSCI17720-24
Discussion
Effects on leaf area and photon capture.
Alternating R/B photons did not increase growth or leaf area at either DLI with a 24-hr photoperiod (Figs. 2, 4, and 6). At a high DLI of 23 mol⋅m−2⋅d−1, the leaf area under alternating R/B photons was 15% higher than of plants grown under concurrent R+B photons, but the difference was not statistically significant (Fig. 4B). There was a statistically significant decrease in the NAR (15.9%) under alternating R/B photons (Fig. 4C), which was contrary to the findings of Ohtake et al. (2018). The 15.9% decrease in the NAR offset the effect of the higher leaf area and photon capture under alternating R/B photons and resulted in a similar dry mass (Fig. 4).
At a lower DLI of 8.6 mol⋅m−2⋅d−1, alternating R/B photons resulted in a lower dry mass for ‘Grand Rapids’ and ‘Rex’ compared with concurrent R+B photons (Fig. 6A). The difference in dry mass, closely followed by the difference in the leaf area (Fig. 6A and 6B), indicated that the change in dry mass was primarily driven by the change in the leaf area and, thus, photon capture. Except for that of ‘Rex’, the NAR did not differ under different light treatments (Fig. 6C). The NAR of ‘Rex’ grown under a DLI of 8.6 mol⋅m−2⋅d−1 was highest under concurrent R+B photons, similar to our findings under DLI of 23 mol⋅m−2⋅d−1 (Fig. 4C); however, this contradicted the findings of Ohtake et al. (2018).
The underlying mechanism of the increased leaf area and photon capture under alternating R/B photons observed during previous studies is not clear. The data do not confirm that the larger leaf area was a result of interactions between signaling pathways of phytochrome (R/far-R light photoreceptor) and cryptochrome (B light photoreceptor) (Chen et al. 2017; Masuda et al. 2021; Ohtake et al. 2021).
Potential mediation by phytochrome.
Phytochromes are known to meditate the shade-avoidance response, including stem elongation (Craver et al. 2018; Hitz et al. 2019), early flowering (Zhang et al. 2020), and leaf expansion (Craver et al. 2018; Park and Runkle 2017). The R photons shift the phytochrome to the active form (Pfr), which inhibits the shade-avoidance response; however, far-red photons convert Pfr to the inactive form (Pr), thus inducing the shade-avoidance response. The dynamic balance between Pr and Pfr is often quantified as phytochrome photo-equilibrium (PPE; ratio of the Pfr to the total phytochrome pool), with low PPE often triggering shade-avoidance responses in plants (Casal 2013; Gommers et al. 2013; Ruberti et al. 2012). Conversely, cryptochrome is the UVA and blue light photoreceptor and activates the signaling pathway antagonistic to the shade-avoidance response. High B photons activate cryptochrome, thus inhibiting hypocotyl elongation, stem elongation, and leaf expansion in Arabidopsis thaliana and horticultural crops including lettuce (Meng and Runkle 2019; Son and Oh 2013; Stutte et al. 2009; Wang and Lin 2020; Wollaeger and Runkle 2015).
However, many recent studies have shown that pure B photons can also elicit the shade-avoidance response and promote stem elongation and canopy expansion. In horticultural food crops, pure B photons promoted stem and/or hypocotyl elongation and, on some occasions, leaf expansion in eggplant (Hirai et al. 2006), sesame (Hata et al. 2013), tomato (Kim et al. 2014), cucumber (Hernández and Kubota 2016), arugula (Johnson et al. 2020; Kong et al. 2019, 2020), cabbage (Kong et al. 2019, 2020), kale (Kong et al. 2019, 2020), mustard (Johnson et al. 2020), sunflower (Vinterhalter et al. 2022), and lettuce (Spalholz et al. 2020). It has been proposed that the elongation response of plants to pure B photons was similar to the shade-avoidance response and was associated with low PPE of pure B photons (Kong et al. 2018; Kong and Zheng 2022). Studies of Arabidopsis also showed that low cryptochrome 1 activity and, to a lesser extent, phototropin are also involved in the shade-avoidance responses under pure B photons (Kong and Zheng 2020, 2022). In cucumber and several microgreen species, the shade-avoidance response elicited by pure B photons increased shoot dry mass compared with pure R photons (Hernández and Kubota 2016; Johnson et al. 2020; Kong et al. 2020). Not surprisingly, the effectiveness of pure B photons to induce elongation growth varied among species (Kong et al. 2018) and even among cultivars within the same species (Hata et al. 2013). In sunflower, which is a high-light plant, pure green, R, and yellow photons, more so than just pure B photons, promoted hypocotyl elongation (Vinterhalter et al. 2022). Notably, it was also shown that concurrent photons of two colors suppressed hypocotyl elongation, but that the presence of R photons was required in the concurrent photons (Vinterhalter et al. 2022). In other words, the combination of green and B photons failed to suppress hypocotyl elongation (Vinterhalter et al. 2022). These results validate the important role of the phytochrome signaling pathway in shade responses and indicate that the shade responses are species-specific.
Effects of pure B photons in leaf expansion.
The RER of leaves during the B period was 61.2% higher than that during the R period, despite the same PPFD of 100 μmol⋅m−2⋅s−1 (Fig. 7A). This indicates that pure B photons induced elongation growth during our study. The PPE of pure B photons was 0.68, which was lower than the PPE of pure R photons and that of concurrent R+B photons (0.88 and 0.87). Internal PPE is an improved metric that accounts for spectral distortion within leaves (Kusuma and Bugbee 2021). The internal PPE for pure B was 0.68, and those for R and concurrent R+B were 0.86 and 0.86. The lower PPE and internal PPE values suggested that pure B photons may act through a phytochrome response because lower values indicate shade. The lower PPE of pure B light could induce leaf elongation and canopy expansion through the phytochrome signaling pathway.
The lower RER during the R period compensated for the higher RER during the B period and did not result in greater leaf and canopy expansion overall because the total leaf area at harvesting was similar to or less than that of lettuce grown under concurrent R+B photons at the same PPFD (Fig. 6B). The higher leaf area and canopy size reported by previous studies (Ohtake et al. 2018; Ohtake et al. 2021) would possibly be the result of elongation growth under pure B photons. However, to compensate for the slower elongation during the R period, elongation of lettuce plants under pure B photons in the previous studies would have been stronger than that in our study. This cultivar-specific response was noted in sesame (Hata et al. 2013).
Pure B photons did not result in the increased yield of lettuce in many previous studies. A previous study showed that pure B photons ranging from 20 to 150 μmol⋅m−2⋅s−1 promoted stem elongation of ‘Kokuyo’ eggplant seedlings but inhibited stem elongation of ‘Okayama-saradana’ lettuce seedlings (Hirai et al. 2006). In the studies conducted by Masuda et al. (2021) and Takasu et al. (2019), lettuce plants were grown under alternating R/B photons with increasing hours of R photons from 0 to 24 h (Fig. 1). Those studies only observed a linear increase in both shoot dry mass and leaf area with the decreasing proportion of B photons without any extension growth under pure B photons (Masuda et al. 2021; Takasu et al. 2019). A study of ‘Green Oakleaf’ and ‘Red Oakleaf’ showed that plants grown under pure B photons had a higher leaf area than plants grown under concurrent R/B photons, but they did not have a higher shoot dry mass (Spalholz et al. 2020). These seemingly contradictory results might be explained by differences in cultivar-specific shade responses.
Effects on photosynthesis.
The Pn of the same lettuce leaves under 100 μmol⋅m−2⋅s−1 R photons was higher than the Pn under 100 μmol⋅m−2⋅s−1 B photons (Fig. 7B). The Pn of lettuce under concurrent R+B photons was intermediate between the Pn of lettuce grown under alternating R/B photons at the same PPFD (Fig. 7B). The lower Pn under B photons was likely a result of competitive absorption of flavonoids and carotenoids against chlorophyll (Liu and van Iersel 2021). The average Pn over 24 h was similar for lettuce plants that were grown under alternating R/B photons and under concurrent R+B photons (Fig. 7B) and resulted in a similar NAR at harvest (Fig. 6C). In summary, both canopy expansion and photosynthesis of lettuce grown under alternating R/B photons of 100 μmol⋅m−2⋅s−1 exhibited different patterns during the R period and B period; however, the overall growth was not higher than the growth of lettuce grown under concurrent R+B photons at 100 μmol⋅m−2⋅s−1 in our study.
Relationship between short-term and long-term measurements of photosynthesis.
The leaf Pn quantifies the instantaneous photosynthetic rate of a small leaf section, whereas the NAR in our study represents the rate of net dry mass production through the whole growth cycle (Snowden et al. 2016). The leaf Pn often does not correlate with dry mass productivity or crop yield (Zelitch 1982). This lack of correlation can be attributed to changes in the carbon use efficiency (CUE) of plants, which is the ability to convert carbohydrates assimilated by photosynthesis into dry mass (van Iersel 2003). The CUE varies widely during different developmental stages, but it is consistent among species (Gifford 1994; McCree and Troughton 1966; van Iersel 2003; van Iersel and Seymour 2002). In our study, the close correlation between the leaf Pn and NAR (Fig. 8) suggested that we selected representative leaves for our leaf Pn measurements, and that the CUE of our lettuce plants did not change dramatically through the whole growth cycle and were not significantly affected by our light treatments.
Conclusions
These studies failed to confirm the findings if previous studies in which alternating R/B light increased lettuce yield compared with concurrent R+B light. Under a DLI at 8.6 mol⋅m−2⋅d−1, concurrent R+B light resulted in the highest total leaf area and shoot dry mass of ‘Grand Rapids’ and ‘Rex’ lettuce (Fig. 6). However, under a higher DLI of 23 mol⋅m−2⋅d−1, no differences in the total leaf area and shoot dry mass grown under concurrent R+B light and alternating R/B light with the same photoperiod were detected (Fig. 4). The NAR of plants grown under alternating R/B photons were either not different or lower than those of plants grown under concurrent R+B photons (Figs. 4C and 6C). We observed a higher canopy RER under pure B photons than under pure R photons at the same PPFD (Fig. 7). This indicated that previously reported benefits of alternating R/B photons on canopy expansion might be the result of higher RER under pure B photons. In contrast, the similar leaf area despite the higher RER under B photons suggested that the enhancement effect of alternating R/B photons might be cultivar-specific.
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